Summary of Work: Pneumocystis carinii is a major opportunistic pathogen of immunocompromised patients. Because P. carinii cannot be cultured, molecular approaches have been utilized to identify and characterize antigens of this organism. Recombinant antigens can then be used to examine host immune responses to P. carinii infections. We have an ongoing project to characterize the antigens of P. carinii in both rat and human infections. We have previously purified the major surface glycoprotein (MSG) of both rat and human pneumocystis using HPLC. It is necessary to use P. carinii from both sources because they are different antigenically; specifically, the major surface antigen in rat and human P. carinii are clearly different. Subsequently, we identified a number of clones from a cDNA library of rat P. carinii that contain genes encoding for the MSG. These clones are clearly related but not identical, demonstrating that multiple genes encode the MSU. Over the past year we have continued studies to characterize genes of potential antigens of P. carinii. We have cloned a number of human P. carinii MSG genes and have produced a recombinant fragment encoding a highly conserved region of human P. carinii MSGs that can be expressed at high levels. Preliminary studies have demonstrated that most humans have antibodies to the recombinant peptide, supporting the hypothesis that most humans have been previously infected with P. carinii. We are also studying the region upstream of the gene, which presumably plays a role in MSG expression, for potential promoter activity. In addition, we have begun studies to characterize other P. carinii antigens. We have identified a family of genes encoding proteases related to the yeast kexin, which functions as a pro-protein processing enzyme. This protease appears to be expressed on the surface of P. carinii. The goal of this study is to better understand the pathogenesis of P. carinii pneumonia, with the hope that we can use this information to control or prevent this disease.